RNAi modulation of Aha and therapeutic uses thereof

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of an Aha gene (Aha1 gene), comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of an Aha gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier, methods for treating diseases caused by Aha1 expression and the expression of an Aha gene using the pharmaceutical composition; and methods for inhibiting the expression of an Aha gene in a cell.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/801,840, filed May 19, 2006, which is hereby incorporated byreference in its entirely.

GOVERNMENT SUPPORT

The work described herein was carried out, at least in part, using fundsfrom the United States government provided under the National Institutesof Health Grant No. GM 42336 and GM 45678/NIH RR 11823. The U.S.government may therefore have certain rights in the invention.

FIELD OF THE INVENTION

The present invention concerns methods of treatment using modulators ofthe gene Activator of Beat Shock Protein 90 ATPase (Aha). Morespecifically, the invention concerns methods of treating disordersassociated with undesired Aha activity, by administering shortinterfering RNA which down-regulate the expression of Aha, and agentsuseful therein.

BACKGROUND OF THE INVENTION

Activator of Heat Shock Protein 90 ATPase 1 (herein: Aha1) is anactivator of the ATPase-activity of Hsp90 and is able to stimulate theinherent activity of yeast Hsp90 by 12-fold and human Hsp90 by 50-fold(Panaretou, B., et al., Mol. Cell 2002, 10:1307-1318). Biochemicalstudies have shown that Aha1 binds to the middle region of Hsp90(Panaretou et al. 2002, supra, Lotz, G. P., et al., J. Biol. Chem. 2003,273; 17228-17235), and recent structural studies of the Aha1-Hsp90 corecomplex suggest that the co-chaperone promotes a conformational switchin the middle segment catalytic loop (370-390) of Hsp90 that releasesthe catalytic Arg380 and facilitates its interaction with ATP in theN-terminal nucleotide-binding domain (Meyer, P., et al., EMBO J. 2000,23:511-519).

The molecular chaperons Heat shock protein 90 (Hsp90) is responsible forthe in vivo activation or maturation of specific client proteins(Picard, D., Cell Mol. Life Sci. 2002, 59:1640-1648; Pearl, L. H., andProdromou, C., Adv. Protein Chem. 2002, 59:157-185; Pratt, W. B., andToft, D. O., Exp. Biol. Med. 2003, 228-111-133; Prodromou, C., andPearl, L, H., Curr. Cancer Drug Targets 2003, 3:301-323). Crucial tosuch activation is the essential ATPase activity of Hsp90 (Panaretou,B., et al, EMBO J. 1998, 17:4829-4836), which drives a conformationalcycle involving transient association of the N-terminalnucleotide-binding domains within the Hsp90 dimer (Prodromou, C., et al,EMBO J. 2000, 19:4383-4392).

As a molecular chaperone, HSP90 promotes the maturation and maintainsthe stability of a large number of conformationally labile clientproteins, most of which are involved in biologic processes that areoften deranged within tumor cells, such as signal transduction,cell-cycle progression and apoptosis. As a result, and in contrast toother molecular targeted therapeutics, inhibitors of HSP90 achievepromising anticancer activity through simultaneous disruption, of manyoncogenic substrates within cancer cells (Whitesell L, and Dai C.,Future Oncol. 2005; 1:529-540; WO 03/067262). Furthermore, HSP90 hasbeen implicated in the degradation of Cystic Fibrosis TransmembraneConductance Regulator (CFTR), Mutations in the CFTR gene lead todefective folding and ubiquination of the protein as a consequence ofHSP90 ATPase activity. Following ubiquitination, CFTR is degraded beforeit can reach its site of activity. Lack of active CFTR then leads to thedevelopment of cystic fibrosis in human subjects having such mutation.Therefore, the inhibition of HSP90 activity may be beneficial forsubjects suffering from cancer or Cystic Fibrosis.

Hsp90 constitutes about 1-2% of total cellular protein (Pratt, W. B.,Annu. Rev. Pharmacol Toxicol. 1997, 37:297-326), and the inhibition ofsuch large amounts of protein by means of an antagonist or inhibitorwould potentially require the introduction of excessive amounts of theinhibitor or antagonist into a cell. An alternative approach is theinhibition of activators of HSP90's ATPase activity, such as Anal, whichare present in smaller amounts. By downregulating the amount of Aha1present in the cell the activity of HSP90 may be lowered substantially.

Significant sequence homology exists between Homo sapiens(NM_(—)01211.1), Mus musculus (NM_(—)146036.1) and Pan troglodytes(XM_(—)510094.1) Aha 1. A clear rattus norvegicus homologue of Aha 1 hasnot been identified; however, there is a Rattus norvegicus(XM_(—)223680.3) gene which has been termed activator of heat shockprotein. ATPase homolog 2 (Ahsa 2) on the basis of its sequence homologyto yeast Ahsa 2. Its sequence is homologous to mus musculus RIKEN cDNA1110064P04 gene (NM_(—)172391.3), which is in turn similar in sequenceto Aus musculus Aha 1 except for N-terminal truncation. A homo sapiensAhsa 2 (NM_(—)152392.1) has also been predicted, but sequence homologyis limited. The functions of these latter three genes have not beensufficiently elucidated. However, there exists one region in which allof the above sequences are identical, and which may be used as thetarget for RNAi agents. It may be advantageous to inhibit the activityof more than one Aha gene.

Recently, double-stranded RNA molecules (dsRNA) have been shown to blockgene expression in a highly conserved regulatory mechanism known as RNAinterference (RNAi), WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

Despite significant advances in the field of RNAi and advances in thetreatment of pathological processes mediated by HSP90, there remains aneed for an agent that can selectively and efficiently attenuate HSP90ATPase activity using the cell's own RNAi machinery. Such agent shallpossess both high biological activity and in vivo stability, and shalleffectively inhibit expression of a target Aha gene, such as Aha1, foruse in treating pathological processes mediated directly or indirectlyby Aha expression, e.g. Aha1 expression.

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of an Aha genein a cell or mammal using such dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases mediated by the expression of an Aha gene, such as in cancer orcystic fibrosis. The dsRNA of the invention comprises an RNA strand (theantisense strand) having a region which is less than 30 nucleotides inlength, generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an mRNA transcript of an Aha gene.

In one aspect, the invention provides doable-stranded ribonucleic acid(dsRNA) molecules for inhibiting the expression of an Aha gene. ThedsRNA comprises at least two sequences that are complementary to eachother. The dsRNA comprises a sense strand comprising a first sequenceand an antisense strand comprising a second sequence. The antisensestrand comprises a nucleotide sequence which is substantiallycomplementary to at least part of an mRNA encoding ah Aha gene, and theregion of complementarity is less than 30 nucleotides in length,generally 19-24 nucleotides in length. The dsRNA effects cleavage of anmRNA encoding an Aha gene within the target sequence of a second dsRNAhaving a sense strand chosen front the group of SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ IDNO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 43, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 49,SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO:59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ IDNO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87,SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO:97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQID NO: 107, SEQ ID NO: 109, SEQ ID NO: 1.11, SEQ ID NO: 113, SEQ ID NO:115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO:133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQID NO: 143, SEQ ID NO: 1.45, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQID NO: 181, and SEQ ID NO: 183, and an antisense strand complementary tothe latter sense strand and chosen from the group of SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ IDNO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96,SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ IDNO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114,SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ IDNO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132,SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ IDNO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150,SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ IDNO: 160, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170,SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ IDNO: 180, SEQ ID NO: 182, and SEQ ID NO: 184 (see Table 1 and Table 2).The Aha gene is preferably an Aha1 gene, and more preferably a Homosapiens Aha1 gene. The dsRNA, upon contacting with a cell expressing theAha gene, may is inhibit the expression of the Aha gene in said cell byat least 20%, or at least 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%,70%, 85%, 90% or 95%, e.g. in HeLa and/or MLE 12 cells. The dsRNA may bedifferent from said second dsRNA, but may have at least 5, at least 10,at least 15, at least 18, or at least 20 contiguous nucleotides perstrand in common with one of the above named nucleotide sequences.

Preferably, the second dsRNA is chosen from the group of AL-DP-7301,AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322, AL-DP-7324, AL-DP-7325,AL-DP-7326, AL-DP-7327, AL-DP-7329, AL-DP-7331, AL-DP-7333, AL-DP-7340,AL-DP-7342, AL-DP-7303, AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316,and AL-DP-7337, AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344,AL-DP-7306, AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-73.23, AL-DP-7335,AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328, AL-DP-7330,AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251, AL-DP-9252, AL-DP-9253,AL-DP-9254, AL-DP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259,AL-DP-9260, AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265,AL-DP-9266, AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276, AL-DP-9277,AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-BP-9284,AL-DP-9285, AL-DP-9286, AL-DP-9287, AL-DP-9288, and AL-DP-9289 (seeTable 1 and Table 2).

Alternatively, the dsRNA itself may be chosen from the group ofAL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322, AL-DP-7324,AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329, AL-DP-7331, AL-DP-7333,AL-DP-7340, AL-DP-7342, AL-DP-7303, AL-DP-7305, AL-DP-7307, AL-DP-7309,AL-DP-7316, and AL-DP-7337, AL-DP-7304, AL DP-7312, AL-DP-7339,AL-DP-7344, AL-DP-7306, AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323,AL-DP-7335, AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328,AL-DP-7330, AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251, AL-DP-9252,AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258,AL-DP-9259, AL-DP-9260, AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9264,AL-DP-9265, AL-DP-9266, AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270,AL-DP-9271, AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276,AL-DP-9277, AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282, AL-DP-9283,AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287, AL-DP-9288, andAL-DP-9289 (see Table 1 and Table 2).

The dsRNA may comprise at least one modified nucleotide. Preferably, themodified nucleotide is chosen from the group of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group. Alternatively, the modified nucleotide ischosen from the group of; a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an a basicnucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide,morpholino nucleotide, a phosphoramidate, and a non-natural basecomprising nucleotide.

In another aspect, the invention provides an isolated cell composing oneof the dsRNAs of the invention. The cell is generally a mammalian cell,such as a human cell. Other embodiments of the cell comprising a dsRNAof the invention are as provided for other aspects of the inventionabove.

In yet another aspect, a pharmaceutical composition for inhibiting theexpression of an Aha gene in an organism is provided, comprising a dsRNAand a pharmaceutically acceptable carrier, wherein the dsRNA comprisesat least two sequences that are complementary to each other and whereina sense strand comprises a first sequence and an antisense strandcomprises a second sequence comprising a region of complementarity whichis substantially complementary to at least a part of a mRNA encoding anAha gene, and wherein said region of complementarity is less than 30nucleotides in length, and wherein the dsRNA effects cleavage of an mRNAencoding an Aha gene within the target sequence of a second dsRNA havinga sense strand chosen from the group of SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ IDNO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ IDNO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97,SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ IDNO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115,SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ IDNO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133,SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ IDNO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151,SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ IDNO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171,SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ IDNO: 181, and SEQ ID NO: 183, and an antisense strand complementary tothe latter sense strand and chosen from the group of SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ IDNO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96,SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ CD NO: 104, SEQ IDNO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114,SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ IDNO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132,SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ IDNO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150,SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ IDNO: 160, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170,SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ IDNO: 180. SEQ ID NO: 182, and SEQ ID NO: 184 (see Table 1 and Table 2).Therein, the Aha gene may be an Aha1 gene, and preferably a Homo sapiensAha1 gene. The dsRNA comprised in the pharmaceutical composition may,upon contact with a cell expressing said Aha gene. Inhibit theexpression of said Aha gene in said cell by at least 20%, or at least25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 85%, 90% or 95%, e.g.in HeLa and/or MLE 12 cells. The dsRNA may be different from said seconddsRNA, hot may have at least 5, at least 10, at least 15, at least 18,or at least 20 contiguous nucleotides per strand in common with one ofthe above named nucleotide sequences.

Preferably, the second dsRNA is chosen from the group of AL-DP-7301,AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322, AL-DP-7324, AL-DP-7325,AL-DP-7326, AL-DP-7327, AL-DP-7329, AL-DP-7331, AL-DP-7333, AL-DP-7340,AL-DP-7342, AL-DP-7303, AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316,and AL-DP-7337, AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344,AL-DP-7306, AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328, AL-DP-7330,AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251, AL-DP-9252, AD-DP-9253,AL-DP-9254, AL-DP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259,AL-DP-9260, AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265,AL-DP-9266, AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276, AL-DP-9277,AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-DP-9284,AL-DP-9285, AL-DP-9286, AL-DP-9287, AL-DP-9288, and AL-DP-9289 (seeTable 1 and Table 2).

Alternatively, the dsRNA comprised in the pharmaceutical compositionitself may be chosen from the group of AL-DP-7301, AL-DP-7308,AL-DP-7318, AL-DP-7320, AL-DP-7322, AL-DP-7324, AL-DP-7325, AL-DP-7326,AL-DP-7327, AL-DP-7329, AL-DP-7331, AL-DP-7333, AL-DP-7340, AL-DP-7342,AL-DP-7303, AL-DP-7305, AL-DP-307, AL-DP-7309, AL-DP-7316, andAL-DP-7337, AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344, AL-DP-7306,AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335, AL-DP-7338,AL-BP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328, AL-DP-7330, AL-DP-7336,AL-DP-7348, AL-DP-9250, AL-DP-9251, AL-DP-9252, AL-DP-9253, AL-DP-9254,AL-PP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259, AL-DP-9260,AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-0264, AL-DP-9265, AL-BP-9266,AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271, AL-DP-9272.AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276, AL-DP-9277, AL-DP-9279,AL-DP-9280, AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-DP-9284, AL-DP-9285,AL-DP-9286, AL-DP-9287, AL-DP-9288, and AL-DP-9289 (see Table 1 andTable 2).

The dsRNA comprised in the pharmaceutical composition may comprise atleast one modified nucleotide. Preferably, said modified nucleotide ischosen from the group of; a 2′-O-methyl modified nucleotide, anucleotide comprising a 5′-phosphorothioate group, and a terminalnucleotide linked to a cholesteryl derivative or dodecanoic acidbisdecylamide group. Alternatively, said modified nucleotide is chosenfrom the group of; a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide.

In yet another aspect, a method for inhibiting the expression of an Ahagene in a cell is provided, the method comprising:

(a) introducing into the cell a double-stranded ribonucleic acid(dsRNA), wherein the dsRNA comprises at least two sequences that arecomplementary to each other and wherein a sense strand comprises a firstsequence and an antisense strand comprises a second sequence comprisinga region of complementarity which is substantially complementary to atleast a part of a mRNA encoding Aha1, and wherein said region ofcomplementarity is less than 30 nucleotides in length and wherein thedsRNA effects cleavage of an mRNA encoding an Aha gene within the targetsequence of a second dsRNA having a sense strand chosen from the groupof SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ IDNO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ IDNO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83,SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO:93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ IDNO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111,SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ IDNO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129,SEQ ID NO: 131, SEQ ID NO: 133. SEQ ID NO: 135, SEQ ID NO: 137, SEQ IDNO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147,SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ IDNO: 157, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167,SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ IDNO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 183, and anantisense strand complementary to the latter sense strand and chosenfrom the group of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, SEQ ID NO: 76, SEQ ID NO: 7.8, SEQ ID NO: 80, SEQ ID NO: 82,SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO:92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ IDNO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110,SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ IDNO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128,SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ IDNO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146,SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ IDNO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 166,SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ IDNO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, and SEQ ID NO:184; and(b) maintaining the cell produced in step (a) for a time sufficient toobtain degradation, of the mRNA transcript, of an Aha gene, therebyinhibiting expression of an Aha gene in the cell. The Aha gene ispreferably an Aha1 gene, and more preferably a homo sapiens Aha1 gene.The dsRNA may be different from said second dsRNA, but may have at least5, at least 10, at least 15, at least 18, or at least 20 contiguousnucleotides per strand In common with one of the above named nucleotidesequences.

Preferably, the second dsRNA is chosen from the group of AL-DP-7301,AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322, AL-DP-7324, AL-DP-7325,AL-DP-7326, AL-DP-7327, AL-DP-329, AL-DP-7331, AL-DP-7333, AL-DP-7340,AL-DP-7342, AL-DP-7303, AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316,and AL-DP-7337, AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344,AL-DP-7306, AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7333,AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328, AL-DP-7330,AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251, AL-DP-9252, AL-DP-9253,AL-DP-9254, AL-DP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259,AL-DP-9260, AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265,AL-DP-9266, AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276, AL-DP-9277,AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-DP-9234,AL-DP-9285, AL-DP-9286, AL-DP-9287, AL-DP-9288, and AL-DP-9289 (seeTable 1 and Table 2).

Alternatively, the dsRNA itself is chosen from the group of AL-DP-7301,AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322, AL-DP-7324, AL-DP-7325,AL-DP-7326, AL-DP-7327, AL-DP-7329, AL-DP-7331, AL-DP-7333, AL-DP-7340.AL-DP-7342, AL-DP-7303, AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316,and AL-DP-7337, AL-DP-7304, AL-DP-7312, AL-DP-339, AL-DP-7344,AL-DP-7306, AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328, AL-DP-7330,AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251, AL-DP-9252, AL-DP-9253,AL-DP-9254, AL-DP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259,AL-DP-9260, AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265,AL-DP-9266, AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276, AL-DP-9277,AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-DP-9284,AL-DP-9285, AL-DP-9286, AL-DP-9287, AL-DP-9283, and AL-DP-9289.Preferably, the method is performed in vitro. Other embodiments of themethod for inhibiting the expression of an Aha gene in a cell are asprovided for other aspects of the invention above.

In yet another aspect, a method of treating, preventing or managingpathological processes mediated by Aha expression is provided,comprising administering to a patient in need of such treatment,prevention or management a therapeutically or prophylactically effectiveamount of a dsRNA, wherein the dsRNA comprises at least two sequencesthat are complementary to each other and wherein a sense strandcomprises a first sequence and an antisense strand comprises a secondsequence comprising a region of complementarity which is substantiallycomplementary to at least a part of a mRNA encoding Aha1, and whereinsaid region of complementarity is less than 30 nucleotides in length andwherein the dsRNA effects cleavage of an mRNA encoding an Aha genewithin the target sequence of a second dsRNA having a sense strandchosen from the group of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9. SEQID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19,SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ IDNO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ IDNO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99,SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ IDNO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117,SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ IDNO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135,SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ IDNO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153,SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 103, SEQ IDNO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173,SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQID NO: 183, and an antisense strand complementary to the latter sensestrand and chosen from the group of SEQ ID NO: 6, SEQ ID NO: 8, SEQ IDNO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ IDNO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70,SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ IDNO: 90. SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO:108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:182, and SEQ ID NO: 184. The dsRNA may be different from said seconddsRNA, but may have at least 5, at least 10, at least 15, at least 18,or at least 20 contiguous nucleotides per strand in common with one ofthe above named nucleotide sequences.

Preferably, the second dsRNA is chosen from the group of AL-DP-7301,AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322, AL-DP-7324, AL-DP-7325,AL-DP-7326, AL-DP-7327, AL-DP-7329, AL-DP-7331, AL-DP-7333, AL-DP-7340,AL-DP-7342, AL-DP-7303, AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316,and AL-DP-7337, AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344,AL-DP-7306, AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328, AL-DP-7330,AL-DP-7336, AL-DP-7345, AL-DP-9250, AX-DP-9251, AL-DP-9252, AL-DP-9253,AL-DP-9254, AL-DP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259,AL-DP-9260, AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265,AL-DP-9266, AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276, AL-DP-9277,AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-DP-9284,AL-DP-9285, AL-DP-9286, AL-DP-9287, AL-DP-9288, and AL-DP-9289 (seeTable 1 and Table 2).

Alternatively, the dsRNA itself is chosen from the group of AL-DP-7301,AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322, AL-DP-7324, AL-DP-7325,AL-DP-7326, AL-DP-7327, AL-DP-7329, AL-DP-7331, AL-DP-7333, AL-DP-7340,AL-DP-7342, AL-DP-7303, AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316,and AL-DP-7337, AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344,AL-DP-7306, AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328, AL-DP-7330,AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251, AL-DP-9252, AL-DP-9253,AL-DP-9254, AL-DP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259,AL-DP-9260, AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265,AL-DP-9266, AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276, AL-DP-9277,AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-DP-9284,AL-DP-9285, AL-DP-9286, AL-DP-9287, AL-DP-9288, and AL-DP-9289. Otherembodiments of the method comprising administering a dsRNA of theinvention are as provided for other aspects of the invention above.

In yet another aspect, a vector for inhibiting the expression of an Ahagene in a cell is provided, said vector comprising a regulatory sequenceoperably linked to a nucleotide sequence that encodes at least onestrand of a dsRNA, wherein one of the strands of said dsRNA issubstantially complementary to at least a part of a mRNA encoding Aha1and wherein said dsRNA is less than 30 base pairs in length and wherein,the dsRNA effects cleavage of an mRNA encoding an Aha gene within thetarget sequence of a second dsRNA having a sense strand chosen from thegroup of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: % SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 15. SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO:45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ IDNO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO:83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ IDNO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:11.1, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119,SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ IDNO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137,SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ IDNO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155,SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 165, SEQ IDNO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175,SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 183, andan antisense strand complementary to the latter sense strand and chosenfrom the group of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82,SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO:92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ IDNO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110,SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ IDNO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128,SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ IDNO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146,SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ IDNO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 166,SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ IDNO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, and SEQ ID NO:184. The dsRNA may be different from said second dsRNA, bid may have atleast 5, at least 10, at least 15, at least 18, or at least 20contiguous nucleotides per strand in common with one of the above namednucleotide sequences.

Preferably, the second dsRNA is chosen from the group of AL-DP-7301,AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322, AL-DP-7324, AL-DP-7325,AL-DP-7326, AL-DP-7327, AL-DP-7320, AL-DP-7331, AL-DP-7333, AL-DP-7340,AL-DP-7342, AL-DP-7303, AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316,and AL-DP-7337, AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344,AL-DP-7306, AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328, AL-DP-7330,AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251, AL-DP-9252, AL-DP-9253,AL-DP-9254, AL-DP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259,AL-DP-9260, AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265,AL-DP-9266, AL-DP-926, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276, AL-DP-9277,AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-DP-9284,AL-DP-9285, AL-DP-9286, AL-DP-9287, AL-DP-9288, and AL-DP-9289 (seeTable 1 and Table 2). Other embodiments of the vector of the inventionare as provided for other aspects of the invention above.

In yet another aspect, an isolated cell comprising the above vector isprovided. Other embodiments of the cell comprising a vector of theinvention are as provided for other aspects of the invention above.

TABLE 1 RNAi agents for the down-regulation of homo sapiens(NM_012111.1), mus musculus (NM_146036.1) and pan troglodytes(XM_510094.1) Aha 1, and minimal off- target interactions in rat cells;AL-DP-7561, AL-DP-7562, AL-DP-7563 and AL-DP-7564 are additionallycross-reactive to mus musculus (NM_172391.3) and rattus norvegicus(XM_223680.3) Aha 2 SEQ SEQ Duplex ID Antisense strand ID identifierSense strand sequence¹ NO: sequence¹ NO: AL-DP-7299auugguccacggauaagcuTT 1 agcuuauccguggaccaauTT 2 AL-DP-7300gugaguaagcuugauggagTT 3 cuccaucaagcuuacucacTT 4 AL-DP-7301agucaaaauccccacuuguTT 5 acaaguggggauuuugacuTT 6 AL-DP-7302aaaucucguggccuuaaugTT 7 cauuaaggccacgagauuuTT 8 AL-DP-7303gagauuagugugagccuugTT 9 caaggcucacacuaaucucTT 10 AL-DP-7304aaucucguggccuuaaugaTT 11 ucauuaaggccacgagauuTT 12 AL-DP-7305agauuagugugagccuugcTT 13 gcaaggcucacacuaaucuTT 14 AL-DP-7306cgggcggacgccaccaacgTT 15 cguugguggcguccgcccgTT 16 AL-DP-7307ggcggacgccaccaacgtcTT 17 gacguugguggcguccgccTT 18 AL-DP-7308gggcggacgccaccaacguTT 19 acguugguggcguccgcccTT 20 AL-DP-7309caacgucaacaacuggcacTT 21 gugccaguuguugacguugTT 22 AL-DP-7310gcgggcggacgccaccaacTT 23 guugguggcguccgcccgcTT 24 AL-DP-7311aucucguggccuuaaugaaTT 25 uucauuaaggccacgagauTT 26 AL-DP-7312acgucaacaacuggcacugTT 27 cagugccaguuguugacguTT 28 AL-DP-7313accaacgucaacaacuggcTT 29 gccaguuguugacguugguTT 30 AL-DP-7314acgcuggaucguggaggagTT 31 cuccuccacgauccagcguTT 32 AL-DP-7315agacccacgcuggaucgugTT 33 cacgauccagcgugggucuTT 34 AL-DP-7316gacccacgcuggaucguggTT 35 ccacgauccagcgugggucTT 36 AL-DP-7317gaauuuacaucagcacccuTT 37 agggugcugauguaaauucTT 38 AL-DP-7318gggaauuuacaucagcaccTT 39 ggugcugauguaaauucccTT 40 AL-DP-7319ugggaauuuacaucagcacTT 41 gugcugauguaaauucccaTT 42 AL-DP-7320ccaacgucaacaacuggcaTT 43 ugccaguuguugacguuggTT 44 AL-DP-7321aaguggggugagggagaccTT 45 ggucucccucaccccacuuTT 46 AL-DP-7322acacaaaucucguggccuuTT 47 aaggccacgagauuuguguTT 48 AL-DP-7323acccacgcuggaucguggaTT 49 uccacgauccagcguggguTT 50 AL-DP-7324gagucaaaauccccacuugTT 51 caaguggggauuuugacucTT 52 AL-DP-7325gagcucuauagaguguuuaTT 53 uaaacacucuauagagcucTT 54 AL-DP-7326ggcagcgguacuacuuugaTT 55 ucaaaguaguaccgcugccTT 56 AL-DP-7327gacacaaaucucguggccuTT 57 aggccacgagauuugugucTT 58 AL-DP-7328agcgggcggacgccaccaaTT 59 uugguggcguccgcccgcuTT 60 AL-DP-7329caaaauccccacuuguaagTT 61 cuuacaaguggggauuuugTT 62 AL-DP-7330gagacccacgcuggaucguTT 63 acgauccagcgugggucucTT 64 AL-DP-7331gagccuugccaaagaugagTT 65 cucaucuuuggcaaggcucTT 66 AL-DP-7332ugacacaaaucucguggccTT 67 ggccacgagauuugugucaTT 68 AL-DP-7333ggagcucuauagaguguuuTT 69 aaacacucuauagagcuccTT 70 AL-DP-7334cccacgcuggaucguggagTT 71 cuccacgauccagcgugggTT 72 AL-DP-7335gauccccaauuugucugauTT 73 aucagacaaauuggggaucTT 74 AL-DP-7336gagauccccaauuugucugTT 75 cagacaaauuggggaucucTT 76 AL-DP-7337agccugacacaaaucucguTT 77 acgagauuugugucaggcuTT 78 AL-DP-7338agauccccaauuugucugaTT 79 ucagacaaauuggggaucuTT 80 AL-DP-7339agggagacccacgcuggauTT 81 auccagcgugggucucccuTT 82 AL-DP-7340gagggagacccacgcuggaTT 83 uccagcgugggucucccucTT 84 AL-DP-7341gccaaguggggugagggagTT 85 cucccucaccccacuuggcTT 86 AL-DP-7342uggcagcgguacuacuuugTT 87 caaaguaguaccgcugccaTT 88 AL-DP-7343ugagggagacccacgcuggTT 89 ccagcgugggucucccucaTT 90 AL-DP-7344aguggagauuagugugagcTT 91 gcucacacuaaucuccacuTT 92 AL-DP-7345aggagcucuauagaguguuTT 93 aacacucuauagagcuccuTT 94 AL-DP-7346agcgguacuacuuugagggTT 95 cccucaaaguaguaccgcuTT 96 AL-DP-7561cgcuggaucguggaggagcTT 97 gcuccuccacgauccagcgTT 98 AL-DP-7562gcuggaucguggaggagcgTT 99 cgcuccuccacgauccagcTT 100 AL-DP-7563cuggaucguggaggagcggTT 101 ccgcuccuccacgauccagTT 102 AL-DP-7564uggaucguggaggagcgggTT 103 cccgcuccuccacgauccaTT 104 ¹Capital letters= desoxyribonucleotides; small letters = ribonucleotides

TABLE 2 RNAi agents for the down-regalation of homo sapiens(NM_012111.1), mus musculus (NM_146036.1) and pan troglodytes(XM_510094.1) Aha 1, and minimal off- target interactions in human cellsSEQ SEQ Duplex ID Antisense strand ID identifier Sense strand sequence¹NO: sequence¹ NO: AL-DP-9250 gccugacacaaaucucgugTT 105cacgagauuugugucaggcTT 106 AL-DP-9251 ccugacacaaaucucguggTT 107ccacgagauuugugucaggTT 108 AL-DP-9252 acgccaccaacgucaacaaTT 109uuguugacguugguggcguTT 110 AL-DP-9253 agcucuauagaguguuuacTT 111guaaacacucuauagagcuTT 112 AL-DP-9254 gggcuggcagcgguacuacTT 113guaguaccgcugccagcccTT 114 AL-DP-9255 cuggcagcgguacuacuuuTT 115aaaguaguaccgcugccagTT 116 AL-DP-9256 ggaugaaguggagauuaguTT 117acuaaucuccacuucauccTT 118 AL-DP-9257 accagaggagcucuauagaTT 119ucuauagagcuccucugguTT 120 AL-DP-9258 aaguggagauuagugugagTT 121cucacacuaaucuccacuuTT 122 AL-DP-9259 gaggagcucuauagaguguTT 123acacucuauagagcuccucTT 124 AL-DP-9260 gggagacccacgcuggaucTT 125gauccagcgugggucucccTT 126 AL-DP-9261 ugagccugacacaaaucucTT 127gagauuugugucaggcucaTT 128 AL-DP-9262 gcggacgccaccaacgucaTT 129ugacguugguggcguccgcTT 130 AL-DP-9263 cggacgccaccaacgucaaTT 131uugacguugguggcguccgTT 132 AL-DP-9264 gaaguggagauuagugugaTT 133ucacacuaaucuccacuucTT 134 AL-DP-9265 cucguggccuuaaugaaggTT 135ccuucauuaaggccacgagTT 136 AL-DP-9266 ucguggccuuaaugaaggaTT 137uccuucauuaaggccacgaTT 138 AL-DP-9267 aaugggaauuuacaucagcTT 139gcugauguaaauucccauuTT 140 AL-DP-9268 ggaauuuacaucagcacccTT 141gggugcugauguaaauuccTT 142 AL-DP-9269 ggagauuagugugagccuuTT 143aaggcucacacuaaucuccTT 144 AL-DP-9270 cacaaaucucguggccuuaTT 145uaaggccacgagauuugugTT 146 AL-DP-9271 acaaaucucguggccuuaaTT 147uuaaggccacgagauuuguTT 148 AL-DP-9272 ggagacccacgcuggaucgTT 149cgauccagcgugggucuccTT 150 AL-DP-9273 ggacgccaccaacgucaacTT 151guugacguugguggcguccTT 152 AL-DP-9274 gaugaaguggagauuagugTT 153cacuaaucuccacuucaucTT 154 AL-DP-9275 gugagccuugccaaagaugTT 155caucuuuggcaaggcucacTT 156 AL-DP-9276 caaugaauggagagucaguTT 157acugacucuccauucauugTT 158 AL-DP-9277 auuagugugagccuugccaTT 159uggcaaggcucacacuaauTT 160 AL-DP-9278 agaugagccugacacaaauTT 161auuugugucaggcucaucuTT 162 AL-DP-9279 uagugugagccuugccaaaTT 163uuuggcaaggcucacacuaTT 164 AL-DP-9280 uuugccaccaucaccuugaTT 165uugaagcaucucucuccguTT 166 AL-DP-9281 acggagagagaugcuucaaTT 167uugaagcaucucucuccguTT 168 AL-DP-9282 cggagagagaugcuucaaaTT 169uuugaagcaucucucuccgTT 170 AL-DP-9283 aaaauccccacuuguaagaTT 171ucuuacaaguggggauuuuTT 172 AL-DP-9284 auccccaauuugucugaugTT 173caucagacaaauuggggauTT 174 AL-DP-9285 ucaaaauccccacuuguaaTT 175uuacaaguggggauuuugaTT 176 AL-DP-9286 aaauccccacuuguaagauTT 177aucuuacaaguggggauuuTT 178 AL-DP-9287 uccccaauuugucugaugaTT 179ucaucagacaaauuggggaTT 180 AL-DP-9288 auggccaaguggggugaggTT 181ccucaccccacuuggccauTT 182 AL-DP-9289 ggagucaaaauccccacuuTT 183aaguggggauuuugacuccTT 184 ¹Capital letters = desoxyribonucleotides;small letters = ribonucleotides

BRIEF DESCRIPTION OF THE FIGURES

No Figures are presented.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides doable-stranded ribonucleic acid (dsRNA) as wellas compositions and methods for inhibiting the expression of an Aha genein a cell or mammal using the dsRNA. The invention also providescompositions and methods for treating pathological conditions anddiseases in a mammal caused by fee expression of an Aha gene usingdsRNA. dsRNA directs the sequence-specific degradation of mRNA through aprocess known as RNA interference (RNAi).

The dsRNA of the invention comprises an RNA strand (the antisensestrand) having a region which is less than 30 nucleotides in length,generally 19-24 nucleotides in length, and is substantiallycomplementary to at least part of an mRNA transcript of an Aha gene. Theuse of these dsRNAs enables the targeted degradation of mRNAs of genesthat are implicated in replication and or maintenance of cancer cells inmammals, and/or in the degradation of misfolded Cystic FibrosisTransmembrane Conductance Regulator (CFTR). Using cell-based and animalassays, the present inventors have demonstrated that very low dosages ofthese dsRNA can specifically and efficiently mediate RNAi, resulting insignificant inhibition of expression of an Aha gene. Thus, the methodsand compositions of the invention comprising these dsRNAs are useful fortreating pathological processes mediated by Aha expression, e.g. cancerand/or cystic fibrosis, by targeting a gene involved in proteindegradation.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of anAha gene, as well as compositions and methods for treating diseases anddisorders caused by the expression of an Aha gene, such as cancer and/orcystic fibrosis. The pharmaceutical compositions of the inventioncomprise a dsRNA having an antisense strand comprising a region ofcomplementarity which is less than 30 nucleotides in length, generally19-24 nucleotides in length, and is substantially complementary to atleast part of an RNA transcript of an Aha gene, together with apharmaceutically acceptable carrier.

Accordingly, certain aspects of the invention, provide pharmaceuticalcompositions comprising the dsRNA of the invention together with apharmaceutically acceptable carrier, methods of using the compositionsto inhibit expression of an Aha gene, and methods of using thepharmaceutical compositions to treat diseases caused by expression of anAha gene.

Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A”, “T” and “U” (irrespective of whether written in capitalor small letters) each generally stand for a nucleotide that containsguanine, cytosine, adenine, thymine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, thymine, and uracil may bereplaced by other moieties without substantially altering the basepairing properties of an oligonucleotide comprising a nucleotide hearingsuch replacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.

As used herein, “Aha gene” refers to Activator of Beat Shock Protein 90ATPase genes, “Aha1” refers to Activator of Heat Shock Protein 90 ATPase1 genes, non-exhaustive examples of which are found under Genbankaccession numbers NM_(—)12111.1 (Homo sapiens), NM_(—)146036.1 (Musmusculus), and XM_(—)510094.1 (Pan troglodytes), “Aha2” refers toputative Activator of Heat Shock Protein 90 ATPase 2 genes, also knownAhsa1, non-exhaustive examples of which may be found under Genbankaccession numbers N_(—)72391.3 (Mus musculus) and XM_(—)223680.3 (Rattusnorvegicus).

As used herein, “target, sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an Aha gene, including mRNA that is a product of RNA processing of aprimary transcription product. The target sequence of any given RNAiagent of the invention means an mRNA-sequence of X nucleotides that istargeted by the RNAi agent by virtue of the complementarity of theantisense strand of the RNAi agent to such sequence and to which theantisense strand may hybridize when brought into contact with the mRNA,wherein X is the number of nucleotides in the antisense strand plus thenumber of nucleotides in a single-stranded overhang of the sense strand,if any.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and farm a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most, relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “Tally complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding Aha1). For example, a polynucleotide iscomplementary to at least apart, of an Aha1 mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding Aha1.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. The two strands forming the duplexstructure may be different portions of one larger RNA molecule, or theymay be separate RNA molecules. Where the two strands are part of onelarger molecule, and therefore are connected by an uninterrupted chainof nucleotides between the 3′-end of one strand and the 5′ end of therespective other strand forming the duplex structure, the connecting RNAchain is referred to as a “hairpin loop”. Where the two strands areconnected covalently by means other than an uninterrupted chain ofnucleotides between the 3′-end of one strand and the 5′ end of therespective other strand forming the duplex structure, the connectingstructure is referred to as a “linker”. The RNA strands may have thesame or a different number of nucleotides. The maximum number of basepairs is the number of nucleotides in the shortest strand of the dsRNAminus any overhangs that are present in the duplex. In addition to theduplex structure, a dsRNA may comprise one or more nucleotide overhangs.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa, “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that has nonucleotide overhang at either end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus. Mostpreferably, the mismatches are located within 6, 5, 4, 3, or 2nucleotides of the 5′ terminus of the antisense strand and/or the 3′terminus of the sense strand.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is past ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of”, in as far as theyrefer to an Aha gene, e.g. an Aha1 gene, herein refer to the at leastpartial suppression of the expression of an Aha gene, e.g. an Aha1 gene,as manifested by a reduction of the amount of mRNA transcribed from anAha gene which may be isolated from a first cell or group of cells inwhich an Aha gene is transcribed and which has or have been treated suchthat the expression of an Aha gene is inhibited, as compared to a secondcell or group of cells substantially identical to the first cell orgroup of cells but which, has or have not been so treated (controlcells). Preferably, the cells are HeLa or MLE 12 cells. The degree ofinhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to Aha genetranscription, e.g. the amount of protein encoded by an Aha gene whichis secreted by a cell, or found in solution after lysis of such cells,or the number of cells displaying a certain phenotype, e.g. apoptosis orcell surface CFTR. In principle, Aha gene silencing may be determined inany cell expressing the target, either constitutively or by genomicengineering, and by any appropriate assay. However, when a reference isneeded in order to determine whether a given dsRNA inhibits theexpression of an Aha gene by a certain degree and therefore isencompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of an Aha gene, e.g. anAha1 gene, is suppressed by at least about 20%, 25%, 35%, or 50% byadministration of the double-stranded oligonucleotide of the invention.In some embodiment, an Aha gene, e.g. an Aha1 gene, is suppressed by atleast about 60%, 70%, or 80% by administration of the double-strandedoligonucleotide of the invention. In some embodiments, an Aha gene, e.g.an Aha1 gene, is suppressed by at least about 85%, 90%, or 95% byadministration of the double-stranded oligonucleotide of the invention.Table 6 provides values for inhibition of Aha1 expression using variousdsRNA molecules of the invention.

As used herein in the context of Aha expression, e.g. Aha1 expression,the tonus “treat”, “treatment”, and the like, refer to relief from oralleviation of pathological processes mediated by Aha expression. In thecontext of the present invention insofar as it relates to any of theother conditions recited herein below (other than pathological processesmediated by Aha expression), the terms “treat”, “treatment”, and thelike mean to relieve or alleviate at least one symptom associated withsuch condition, or to slow or reverse the progression of such condition.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes mediated by Aha expression or an overt symptom ofpathological processes mediated by Aha expression. The specific amountthat is therapeutically effective can be readily determined by ordinarymedical practitioner, and may vary depending on factors known in theart, such as, e.g. the type of pathological processes mediated by Ahaexpression, the patient's history and age, the stage of pathologicalprocesses mediated by Aha expression, and the administration of otheranti-pathological processes mediated by Aha expression agents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed,

Double-Stranded Ribonucleic Acid (dsRNA)

In one embodiment, the invention provides doable-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of an Aha gene,e.g. an Aha1 gene, in a cell or mammal, wherein the dsRNA comprises anantisense strand comprising a region of complementarity which iscomplementary to at least a part of an mRNA formed in the expression ofan Aha gene, e.g. an Aha1 gene, and wherein the region ofcomplementarity is less than 30 nucleotides in length, generally 19-24nucleotides in length. The dsRNA may be identical to one of the dsRNAsshown in Table 1 and Table 2, or it may effect cleavage of an mRNAencoding an Aha gene within the target sequence of one of the dsRNAsshown in Table 1 and Table 2. Preferably, the dsRNA has at least 5, atleast 10, at least 15, at least 18, or at least 20 contiguousnucleotides per strand in common with at least one strand, butpreferably both strands, of one of the dsRNAs shown in Table 1 and Table2. Alternative dsRNAs that target elsewhere in the target sequence ofone of the dsRNAs provided in Table 1 and Table 2 can readily bedetermined using the target sequence and the flanking Aha1 sequence.

The dsRNA comprises two RNA strands that are sufficiently complementaryto hybridize to form a duplex structure. One strand of the dsRNA (theantisense strand) comprises a region of complementarity that issubstantially complementary, and generally fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of an Aha gene, the other strand (the sense strand) comprisesa region which is complementary to the antisense strand, such that thetwo strands hybridize and form a duplex structure when combined undersuitable conditions. Generally, the duplex structure is between 15 and30, more generally between 18 and 25, yet more generally between 19 and24, and most generally between 19 and 21 base pairs in length.Similarly, the region, of complementarity to the target sequence isbetween 15 and 30, more generally between 18 and 25, yet more generallybetween 1.9 and 24, and most generally between 19 and 21 nucleotides inlength. The dsRNA of the invention may further comprise one or moresingle-stranded nucleotide overhang(s). The dsRNA can be synthesized bystandard methods known in fee art as further discussed below, e.g., byuse of an automated DNA synthesizer, such as are commercially availablefrom, for example, Biosearch, Applied Biosystems, inc. In a preferredembodiment, an Aha gene is the human Aha1 gene. In specific embodiments,the first strand of the dsRNA comprises the sense sequences of the RNAiagents AL-DP-7301-AL-DP-7346 and AL-DP-7561-AL-DP-7564 (Table 1), andAL-DP-9250-AL-DP-9289 (Table 2), and the second sequence is selectedfrom the group consisting of the antisense sequences ofAL-DP-7301-AL-DP-7346 and AL-DP-7561 AL-DP-7564 (Table 1), andAL-DP-9250-AL-DP-9289 (Table 2).

In further embodiments, the dsRNA comprises at least one nucleotidesequence selected from the groups of sequences provided above for theRNAi agents AL-DP-7301-AL-DP-7346 and AL-DP-7561-AL-DP-7564 (Table 1),and AL-DP-9250-AL-DP-9289 (Table 2). In other embodiments, the dsRNAcomprises at least two sequences selected from this group, wherein oneof the at least two sequences is complementary to another of the atleast two sequences, and one of the at least two sequences issubstantially complementary to a sequence of an mRNA generated in theexpression of an Aha gene, e.g. an Aha1 gene. Generally, the dsRNAcomprises two oligonucleotides, wherein one oligonucleotide may bedescribed as the sense strand in one of the RNAi agentsAL-DP-7301-AL-DP-7346 and AL-DP-7561-AL-DP-7564 (Table 1), andAL-DP-9250-AL-DP-9289 (Table 2), and the second oligonucleotide may bedescribed as the antisense strand in one of the RNAi agentsAL-DP-7301-AL-DP-7346 and AL-DP-7561-AL-DP-7564 (Table 1), andAL-DP-9250-AL-DP-9289 (Table 2).

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided for the RNAi agentsAL-DP-7301-AL-DP-7346 and AL-DP-7561-AL-DP-7564 (Table 1), andAL-DP-9250-AL-DP-9289 (Table 2), the dsRNAs of the invention cancomprise at least one strand of a length of minimally 21 nt. It can bereasonably expected that shorter dsRNAs comprising one of the sequencesprovided herein for the RNAi agents AL-DP-7301-AL-DP-7346 andAL-DP-7561-AL-DP-7564 (Table 1), and AL-DP-9250-AL-DP-9289 (Table 2),minus only a few nucleotides on one or both ends may be similarlyeffective as compared to the dsRNAs described above. Hence, dsRNAscomprising a partial sequence of at least 15, 16, 17, 13, 19, 20, ormore contiguous nucleotides from one of the sequences of the RNAi agentsAL-DP-7301-AL-DP-7346 and AL-DP-7561-AL-DP-7564 (Table 1), andAL-DP-9250-AL-DP-9289 (Table 2), and differing in their ability toinhibit the expression of an Aha gene, e.g. an Aha1 gene, in a FACSassay as described herein below by not more than 5, 10, 15, 20, 25, or30% inhibition from a dsRNA comprising the full sequence, arecontemplated by the invention.

Further dsRNAs that cleave within the target sequence of the RNAi agentsAL-DP-7301-AL-DP-7346 and AL-DP-7561-AL-DP-7564 (Table 1), andAL-DP-9250-AL-DP-9289 (Table 2), can readily be made using the Aha1 genesequence and the respective target sequence. The RNAi agents provided inTable 1 and Table 2 identify a site in the Aha1 mRNA that is susceptibleto RNAi based cleavage. As such the present invention includes RNAiagents that target within the sequence targeted by one of the agents ofthe present invention. As used herein a dsRNA is said to target within,the sequence of a second dsRNA if the dsRNA cleaves the message anywherewithin the mRNA that is complementary to the antisense strand of thesecond dsRNA. Such a dsRNA will generally have least 5, at least 10, atleast 15, at least 18, or at least 20 contiguous nucleotides from one ofthe sequences provided in Table 1 and Table 2 coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in an mRNA encoding an Aha gene. For example, the 3′-most 15nucleotides of the target sequence of AL-DP-7301 combined with the next6 nucleotides from the target Aha1 gene produces a single strand agentof 21 nucleotides that is based on one of the sequences provided inTable 1 and Table 2.

Preferably, the second dsRNA is chosen from the group of dsRNAs having acertain activity in inhibiting the expression of an Aha gene in asuitable assay, such as the assays described herein. Consequently, incertain preferred embodiments, the second dsRNA is chosen from the groupof AL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322,AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329, AL-DP-7331,AL-DP-7333, AL-DP-7340, AL-DP-7342, AL-DP-7303, AL-DP-7305, AL-DP-7307,AL-DP-7309, AL-DP-7316, and AL-DP-7337, AL-DP-7304, AL-DP-7312,AL-DP-733.9, AL-DP-7344, AL-DP-7306, AL-DP-7317, AL-DP-7346, AL-DP-7310,AL-DP-7323, AL-DP-7335, AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315,AL-DP-7328, AL-DP-7330, AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251,AL-DP-9252, AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256, AL-DP-9257,AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9261, AL-DP-9262, AL-DP-9263,AL-DP-9264, AL-DP-9265, AL-DP-9266, AL-DP-9267, AL-DP-9268, AL-DP-9269,AL-DP-9270, AL-DP-9271, AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275,AL-DP-9276, AL-DP-9277, AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282,AL-DP-9283, AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287, AL-DP-9288,and AL-DP-9289.

The dsRNA of the invention can contain one or more mismatches to thetarget sequence. In a preferred embodiment, the dsRNA of the inventioncontains no more than 3 mismatches, if the antisense strand of the dsRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity, and preferably from the 5′-end. For example, for a 23nucleotide dsRNA strand which is complementary to a region of an Ahagene, the dsRNA generally does not contain any mismatch within thecentral 13 nucleotides. In another embodiment, the antisense strand ofthe dsRNA does not contain any mismatch in the region from positions 1,or 2, to positions 9, or 10, of the antisense strand (counting 5′-3′).The methods described within the invention, can be used to determinewhether a dsRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of an Aha gene. Consideration of theefficacy of dsRNAs with mismatches in inhibiting expression of an Aliagene is important, especially if the particular region ofcomplementarity in an Aha gene is known to have polymorphic sequencevariation within the population.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. Moreover, thepresent inventors have discovered that the presence of only onenucleotide overhang strengthens the interference activity of the dsRNA,without affecting its overall stability. dsRNA having only one overhanghas proven particularly stable and effective in vivo, as well as in avariety of cells, cell culture mediums, blood, and serum. Generally, thesingle-stranded overhang is located at the 3′-terminal end of theantisense strand or, alternatively, at the 3′-terminal end of the sensestrand. The dsRNA may also have a blunt end, generally located at the5′-end of the antisense strand. Such dsRNAs have improved stability andinhibitory activity, thus allowing administration at low dosages, i.e.,less than 5 mg/kg body weight of the recipient per day. Generally, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids of the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al.(Edrs.), John. Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Specific examples of preferred dsRNAcompounds useful in this invention include dsRNAs containing modifiedbackbones or no natural internucleoside linkages. As defined in thisspecification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified dsRNA backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Preferred modified dsRNA backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatoms and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part, from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleotides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other preferred dsRNA mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an dsRNA mimetic that has been shown to have,excellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replacedwith an amide containing backbone, in particular an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Representative U.S. patents that teach the preparation of PNA compoundsinclude, hut are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found in Nielsen et al.,Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are dsRNAs withphosphorothioate backbones and oligonucleotides with heteroatombackbones, and in particular .CH₂.NH.CH₂., .CH₂.N(CH₃).O.CH₂. [known asa methylene (methylimino) or MMI backbone], .CH₂.O.N(CH₃).CH₂.,.CH₂.N(CH₃).N(CH₃).CH₂. and .N(CH₃).CH₂.CH₂. [wherein the nativephosphodiester backbone is represented as .O.P.O.CH₂.] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O-, S-, or N-alkyl: O-, S-, or N-alkenyl; O-, S or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted to C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₂, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred dsRNAs comprise one of the following at the 2′ position:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an dsRNA, or a group for improving thepharmacodynamic properties of an dsRNA, and other substituents havingsimilar properties. A preferred modification includes 2′-methoxyethoxy(2′-O.CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOB) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxygroup. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O.CH₂.O.CH₂.N(CH₂)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

DsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L., ed. John Wiley & Sons.1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown, to increase nucleic acidduplex stability by 0.6-1.2.degree, C. (Sanghvi, Y. S., Crooke, S. T.and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Another modification of the dsRNAs of the invention involves chemicallylinking to the dsRNA one or more moieties or conjugates which enhancethe activity, cellular distribution or cellular uptake of the dsRNA.Such moieties. Include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199,86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let,1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg.Med. Chem. Let, 1993, 3, 2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochemie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNAconjugates include, hut are not limited to, U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603:5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873:5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference.

It is not necessary for all positions is a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of an dsRNA compound. These dsRNAs typically contain atleast one region, wherein the dsRNA is modified so as to confer upon thedsRNA increased resistance to nuclease degradation. Increased cellularuptake, and/or increased binding affinity for the target nucleic acid.An additional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of dsRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter dsRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxydsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86: 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., Ann. N.Y., Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg,Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651: Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al. Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate.

Vector Encoded RNAi Agents

The dsRNA of the invention can also be expressed from recombinant viralvectors intracellularly in vivo. The recombinant viral vectors of theinvention comprise sequences encoding the dsRNA of the invention and anysuitable promoter for expressing the dsRNA sequences. Suitable promotersinclude, for example, the U6 or H1 RNA pol III promoter sequences andthe cytomegalovirus promoter. Selection of other suitable promoters iswithin the skill in the art. The recombinant viral vectors of theinvention can also comprise inducible or regulatable promoters forexpression of the dsRNA in a particular tissue or in a particularintracellular environment. The use of recombinant viral vectors todeliver dsRNA of the invention to cells in vivo is discussed in moredetail below.

dsRNA of the invention can be expressed from a recombinant viral vectoreither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g.,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorswhich express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801,the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2:301-310; Eglitis M A (1988),Biotechniques 6:608-614; Miller A D (1990), Hum Gene Therap. 1:5-14;Anderson W P (1998), Nature 392:25-30; and Rubinson D A et al., Nat.Genet. 33:401-406, the entire disclosures of which are hereinincorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the dsRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector comprising, for example, either the U6 orH1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a methodfor constructing the recombinant AV vector, and a method for deliveringthe vector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20:1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61:3096-3101; Fisher K J et al., (1996), J. Virol, 70:520-532;Samulski R et al. (1989), J. Virol. 63; 3822-3826; U.S. Pat. No.5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No.WO 94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

Pharmaceutical Compositions Comprising dsRNA

In one embodiment, the invention provides pharmaceutical compositionscomprising a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition comprising the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of an Aha gene, such as pathological processesmediated by Aha1 expression. Such pharmaceutical compositions areformulated based on the mode of delivery. One example is compositionsthat are formulated for systemic administration via parenteral delivery.

The pharmaceutical compositions of the invention are administered indosages sufficient to inhibit expression, of an Aha gene. The presentinventors have found that, because of their improved efficiency,compositions comprising the dsRNA of the invention can be administeredat surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogrambody weight of recipient per day is sufficient to inhibit or completelysuppress expression of an Aha gene.

In general, a suitable dose of dsRNA will be in the range of 0.01microgram to 5.0 milligrams per kilogram body weight of the recipientper day, generally in the range of 1 microgram to 1 mg per kilogram bodyweight per day. The pharmaceutical composition may be administered oncedaily, or the dsRNA may be administered as two, three, or more sub-dosesat appropriate intervals throughout the day or even using continuousinfusion or delivery through a controlled release formulation. In thatcase, the dsRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage. The dosage unit canalso be compounded for delivery over several days, e.g. using aconventional sustained release formulation which provides sustainedrelease of the dsRNA over a several day period. Sustained releaseformulations are well known, in the art and are particularly useful forvaginal delivery of agents, such as could be used with the agents of thepresent invention. In this embodiment, the dosage unit contains acorresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on fee basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by Aha expression. Such models are used for in vivo testing ofdsRNA, as well as for determining a therapeutically effective dose.

The present invention also includes pharmaceutical compositions andformulations which include the dsRNA compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical, pulmonary, e.g., by inhalation or insufflation of powders oraerosols, including by nebulizer; intratracheal, intranasal, epidermaland transdermal, oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical, formulations include thosein which the dsRNAs of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl ethanolamine=DOPE,dimyristoylphosphatidyl choline=DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol=DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl=DOTAP and dioleoylphosphatidylethanolamine=DOTMA). DsRNAs of the invention may be encapsulated withinliposomes or may form, complexes thereto, in particular to cationicliposomes. Alternatively, dsRNAs may be complexed to lipids, inparticular to cationic lipids. Preferred fatty acids and esters includebut are not limited arachidonic acid, oleic acid, eicosanoic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which dsRNAs of the invention are administered inconjunction with one or more penetration enhancers, surfactants, andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxyeholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-4-fusidate and sodium glycodihydrofusidate.Preferred, fatty acids include arachidonic acid, undecanoic acid, oleicacid, lauric acid, caprylic acid, capric acid, myristic acid, palmiticacid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsof the invention may be delivered orally, in granular form includingsprayed dried particles, or complexed to form micro or nanoparticles.DsRNA complexing agents include poly-amino acids; polyimines;polyacrylates; polyalkylaerylates, polyoxethanes,polyalkylcyanoacrylates; canonized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S.application Ser. No. 08/886,829 (filed Jul., 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999), each of which is incorporated herein by referencein their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carriers) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising two immiscible liquid phases intimately mixed and dispersedwith each other. In general, emulsions may be of either the water-in-oil(w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finelydivided into and dispersed as minute droplets into a bulk oily phase,the resulting composition is called a water-in-oil (w/o) emulsion.Alternatively, when an oily phase is finely divided into and dispersedas minute droplets into a bulk aqueous phase, the resulting compositionis called an oil-in-water (o/w) emulsion. Emulsions may containadditional components in addition to the dispersed phases, and theactive drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p,199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, inc., New York, N. Y., volume 1,p, 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fate, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, In Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth, of microbes, these formulations often incorporatepreservatives. Commonly used preservatives. Included in emulsion,formulations include methyl, paraben, propyl paraben, quaternaryammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid,and boric acid. Antioxidants are also commonly added to emulsionformulations to prevent deterioration of the formulation. Antioxidantsused may be free radical scavengers such as tocopherols, alkyl gallates,butylated hydroxyanisole, butylated hydroxytoluene, or reducing agentssuch as ascorbic acid and sodium metabisulfite, and antioxidantsynergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have, been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions of dsRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system, of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to livecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p, 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed, spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol tatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO₇₅₀), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The (cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known, in the art. Theaqueous phase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C₈-C₁₂) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C₉-C₁₀glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs, lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390: Ritschel, Meth. Find. Exp.Clin. Pharmacol, 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al. Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci, 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids within the gastrointestinal tract, vagina,buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest, because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs is their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations is thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active Ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents info the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than, complex, formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidyl choline (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean. PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Werner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-Ionic surfactant and cholesterol, Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (He et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(m)1, or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42: Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(m)1, galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(m)1 or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristosylphosphat-idylcholine are disclosed in WO 97/13499(Lim et al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al., (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett, 1990, 268, 235)described experiments demonstrating that Liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysics Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent. No. EP 0 445 131 B1and WO 90704384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes, U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that, the contents of such liposomes may include dsRNA. U.S.Pat. No. 5,665,710 to Rahman et al. describes certain methods ofencapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love etal. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly dsRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer, inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al. Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of dsRNAs through the mucosa isenhanced, in addition to bile salts and fatty acids, these penetrationenhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al.,J. Pharm. Pharmacol, 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁-C₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drag Carrier Systems, 1991, p. 92;Muranishi, Critical Reviews in Therapeutic Drag Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol, 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble, vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium chelate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drag Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed. Mack Publishing Co., Easton, Pa., 1990, pages 782-783;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33: Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashitaet al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent, invention, can be defined as compounds feat remove metallicions from solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1996, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal., U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuck as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which isinert, (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate dsRNA in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4 isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,tale, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Pharmaceutical Compositions for the Delivery to the Respiratory Tract

Another aspect of the invention provides for the delivery of IRNA agentsto the respiratory tract, particularly for the treatment of cysticfibrosis. The respiratory tract includes the upper airways, includingthe oropharynx and larynx, followed by the lower airways, which includethe trachea followed by bifurcations into the bronchi and bronchioli.The upper and lower airways are called the conductive airways. Theterminal bronchioli then divide into respiratory bronchioli which thenlead to the ultimate respiratory zone, the alveoli, or deep lung. Thedeep lung, or alveoli, are the primary target of inhaled therapeuticaerosols for systemic delivery of iRNA agents.

Pulmonary delivery compositions can be delivered by inhalation, by thepatient of a dispersion so that the composition, preferably the iRNAagent, within the dispersion can reach the lung where it can, forexample, be readily absorbed through the alveolar region directly intoblood circulation. Pulmonary delivery can be effective both for systemicdelivery and for localized delivery to treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations; administration by inhalation may be oral and/or nasal.Delivery can be achieved with liquid nebulizers, aerosol-based inhalers,and dry powder dispersion devices. Metered-dose devices are preferred.One of the benefits of using an atomizer or inhaler is that thepotential for contamination is minimized because the devices are selfcontained. Dry powder dispersion devices, for example, deliver drugsthat may be readily formulated as dry powders. An iRNA composition maybe stably stored as lyophilized or spray-dried powders by itself or incombination with suitable powder carriers. The delivery of a compositionfor inhalation can be mediated by a dosing timing element which caninclude a timer, a dose counter, time measuring device, or a timeindicator which when incorporated into the device enables dose tracking,compliance monitoring, and/or dose triggering to a patient duringadministration of the aerosol medicament.

Examples of pharmaceutical devices for aerosol delivery include metereddose inhalers (MDIs), dry powder inhalers (DPIs), and air-jetnebulizers. Exemplary delivery systems by inhalation which can bereadily adapted for delivery of the subject iRNA agents are describedin, for example, U.S. Pat. Nos. 5,756,353; 5,858,784; and PCTapplications WO98/31346; WO98/10796; WO00/27359; WO01/54664;WO02/060412. Other aerosol formulations that may be used for deliveringthe iRNA agents are described in U.S. Pat. Nos. 6,294,153; 6,344,194;6,071,497, and PCT applications WO02/066078; WO02/053190; WO01/60420;WO00/66206. Further, methods for delivering iRNA agents can be adaptedfrom those used in delivering other oligonucleotides (e.g., an antisenseoligonucleotide) by inhalation, such as described in Templin et al.,Antisense Nucleic Acid Drug Dev, 2000, 10:359-68; Sandrasagra et al.Expert Opin Biol Ther, 2001, 1:979-83; Sandrasagra et al., AntisenseNucleic Acid Drag Dev, 2002, 12:177-81.

The delivery of the inventive agents may also involve the administrationof so called “pro-drugs”, i.e. formulations or chemical modifications ofa therapeutic substance that require some form of processing ortransport by systems innate to the subject organism to release thetherapeutic substance, preferably at the site where its action isdesired; this latter embodiment may be used in conjunction with deliveryof the respiratory tract, but also together with other embodiments ofthe present invention. For example, the human lungs can remove orrapidly degrade hydrolytically cleavable deposited aerosols over periodsranging from minutes to hours. In the upper airways, ciliated epitheliacontribute to the “mucociliary escalator” by which particles are sweptfrom the airways toward, the mouth. Pavia, D., “Lung MucociliaryClearance,” in Aerosols and the Lung: Clinical and Experimental Aspects,Clarke, S. W. and Pavia, D. Eds., Butterworths, London, 1984. In thedeep lungs, alveolar macrophages are capable of phagocytosing particlessoon after their deposition. Warheit et al. Microscopy Res. Tech., 26:412-422 (1993); and Brain, J, D., “Physiology and Pathophysiology ofPulmonary Macrophages,” in The Reticuloendothelial System, S. M.Reichard and J. Filkins, Eds., Plenum, New. York., pp. 315-327, 1985.

In preferred embodiments, particularly where systemic dosing with theiRNA agent is desired, the aerosoled iRNA agents are formulated asmicroparticles, Microparticles having a diameter of between 0.5 and tenmicrons can penetrate the lungs, passing through most of the naturalbarriers. A diameter of less than ten microns is required to bypass thethroat; a diameter of 0.5 microns or greater is required to avoid beingexhaled.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage tonus of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when, added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodiumcarboxylmethylcellulose, sorbitol and/or dextran. The suspension mayalso contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more ds RNA agents and (b) one or more otherchemotherapeutic agents which function by a non-RNA interferencemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Railway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-dsRNAchemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions of the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, the dsRNAs of the invention can be administered incombination with other known agents effective in treatment ofpathological processes mediated by Aha expression. In any event, theadministering physician can adjust the amount and timing of dsRNAadministration on the basis of results observed using standard measuresof efficacy known in the art or described herein.

Methods for Treating Diseases Caused by Expression of an Aha Gene

The invention relates in particular to the use of a dsRNA or apharmaceutical composition prepared therefrom for the treatment ofCystic Fibrosis. Owing to the inhibitory effect on Aha1 expression, andsRNA according to the invention or a pharmaceutical compositionprepared therefrom can enhance the quality of life of Cystic Fibrosispatients.

Furthermore, the invention relates to the use of a dsRNA or apharmaceutical composition of the invention aimed at the treatment ofcancer, e.g. for inhibiting tumor growth and tumor metastasis. Forexample, the dsRNA or a pharmaceutical composition prepared therefrommay be used for the treatment of solid tumors, like breast cancer, lungcancer, head and neck cancer, brain cancer, abdominal cancer, coloncancer, colorectal cancer, esophagus cancer, gastrointestinal cancer,glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma,ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma,Wilm's tumor, multiple myeloma and for the treatment of skin cancer,like melanoma, for the treatment of lymphomas and blood cancer. Theinvention further relates to the use of an dsRNA according to theinvention or a pharmaceutical composition prepared therefrom, forinhibiting Aha1 expression and/or for inhibiting accumulation of ascitesfluid and pleural effusion in different types of cancer, e.g., breastcancer, lung cancer, head cancer, neck cancer, brain cancer, abdominalcancer, colon cancer, colorectal cancer, esophagus cancer,gastrointestinal cancer, glioma, liver cancer, tongue cancer,neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, retinoblastoma, Wilm's tumor, multiple myeloma, skin cancer,melanoma, lymphomas and blood cancer. Owing to the inhibitory effect onAha1 expression, an dsRNA according to the invention or a pharmaceuticalcomposition prepared therefrom can enhance the quality of life of cancerpatients.

The invention furthermore relates to the use of an dsRNA or apharmaceutical composition thereof, e.g., for treating Cystic Fibrosisor cancer or for preventing tumor metastasis, in combination with otherpharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed, for treating Cystic Fibrosis orcancer and/or for preventing tumor metastasis. Where the pharmaceuticalcomposition aims for the treatment of Cystic fibrosis, preference isgiven to a combination with daily chest physiotherapy, orally appliedpancreatic enzymes, daily oral or inhaled antibiotics to counter lunginfection, inhaled anti-asthma therapy, corticosteroid tablets, dietaryvitamin supplements, especially A and D, inhalation of Pulmozyme™,medicines to relieve constipation or to improve the activity of theenzyme supplements, insulin for CF-related diabetes, medication forCF-associated Liver disease, and oxygen to help with breathing.

Where the pharmaceutical composition aims for the treatment of cancerand/or for preventing tumor metastasis, preference is given to acombination with radiation therapy and chemotherapeutic agents, such ascisplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin ortamoxifen.

The invention can also be practiced by including with a specific RNAiagent another anti-cancer chemotherapeutic agent, such as anyconventional chemotherapeutic agent. The combination of a specificbinding agent with such other agents can potentiate the chemotherapeuticprotocol Numerous chemotherapeutic protocols will present themselves inthe mind of the skilled practitioner as being capable of incorporationinto the method of the invention. Any chemotherapeutic agent can beused, including alkylating agents, antimetabolites., hormones andantagonists, radioisotopes, as well as natural products. For example,the compound of the invention can be administered with antibiotics suchas doxorubicin and other anthracycline analogs, nitrogen mustards suchas cyclophosphamide, pyrimidine analogs such as 5-fluorouracil,cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives,and the like. As another example, in the ease of mixed tumors, such asadenocarcinoma of the breast, where the tumors includegonadotropin-dependent and gonadotropin-independent cells, the compoundcan be administered in conjunction with leuprolide or goserelin(synthetic peptide analogs of LH-RH). Other antineoplastic protocolsinclude the use of a tetracycline compound with another treatmentmodality, e.g., surgery, radiation, etc., also referred to herein as“adjunct antineoplastic modalities.” Thus, the method of the Inventioncan be employed with such conventional regimens with the benefit ofreducing side effects and enhancing efficacy.

Methods for Inhibiting Expression of an Aha Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of an Aba gene in a mammal. The method comprisesadministering a composition of the invention to the mammal such thatexpression of the target Aha gene, e.g. Aha1, is silenced. Because oftheir high specificity, the dsRNAs of the invention specifically targetRNAs (primary or processed) of the target Aha gene. Compositions andmethods for inhibiting the expression of these Aha genes using dsRNAscan be performed as described elsewhere herein.

In one embodiment, the method comprises administering a compositioncomprising a dsRNA, wherein the dsRNA comprises a nucleotide sequencewhich is complementary to at least apart of an RNA transcript of an Ahagene, e.g. Aha1, of the mammal to be treated. When the organism to betreated is a mammal such as a human, the composition may be administeredby any means known in the art including, but not limited to oral orparenteral routes, including intravenous, intramuscular, subcutaneous,transdermal, airway (aerosol), nasal rectal, vaginal and topical(including buccal and sublingual) administration. In preferredembodiments, the compositions are administered by intravenous infusionor injection.

Unless otherwise defined, all technical and scientific terms used,herein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, suitable methods and materialsare described below. All publications, patent applications, patents, andother references mentioned herein are Incorporated by reference in theirentirety. In ease of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES

Gene Walking of an Aha Gene

siRNA design was carried out to identify siRNAs targeting Aha1. The mRNAsequences of Homo sapiens (NM_(—)012111.1), mus musculus (NM_(—)46036.1)and pan troglodytes (XM_(—)510094.1) Aha 1 were examined by computeranalysis to identify homologous sequences of 19 or 21 nucleotides thatyield RNAi agents cross-reactive between these three species. Of thoseidentified, 48 such sequences were selected for minimal off-targetinteractions in rats (at least 3 mismatches, to any other gene in therat genome, or at least two mismatches to any other gene in the ratgenome, wherein one of said at least two mismatches is located in aposition complementary to position 9 or 10 of the antisense strand ofthe corresponding RNAi agent, counting 5′ to 3′) and the correspondingdsRNAs synthesized for screening (AL-DP-7301-AL-DP-7346, see Table 1),AL-DP-7561, AL-DP-7562, AL-DP-7563 and AL-DP-7564 which are additionallycross-reactive to mus musculus (NM_(—)172391.3) and rattus norvegicus(XM_(—)223680.3) Aha 2, were also synthesized and screened. In addition,a further 40 sequences were selected for minimal predicted off-targetinteractions in humans (at least 3 mismatches to any other gene in thehuman genome, or at least two mismatches to any other gene in the humangenome, wherein one of said at least two mismatches is located in aposition complementary to position 9 or 10 of the antisense strand ofthe corresponding RNAi agent, counting 5′ to 3′) and the correspondingdsRNAs synthesized for screening (AL-DP-9250-AL-DP-9289, see Table 2).17 sequences were identified as belonging to both sets (AL-DP-7301,AL-DP-7304, AL-DP-7305, AL-DP-7307, AL-DP-7310, AL-DP-7312, AL-DP-7315,AL-DP-7316, AL-DP-7317, AL-DP-7323, AL-DP-7324, AL-DP-7332, AL-DP-7336,AL-DP-7337, AL-DP-7338, AL-DP-7342, and AL-DP-7344.

dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the Iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection mid purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol-3′), an appropriately modified solid support was used forRNA synthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature untilcompletion of the reaction was ascertained by TLC. After 19 h thesolution was partitioned with dichloromethane (3×100 mL). The organiclayer was dried with anhydrous sodium sulfate, filtered and evaporated.The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-ammo-hexanoic acid (9.12 g. 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodimide(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.Completion of the reaction was ascertained by TLC. The reaction mixturewas concentrated under vacuum and ethyl acetate was added to precipitatediisopropyl area. The suspension was filtered. The filtrate was washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer was dried over sodium sulfate and concentrated togive the crude product which was purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated under vacuum, water was addedto the residue, and the product was extracted with ethyl acetate. Thecrude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethyl amine (3.87 g, 5.2 mL, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (103 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 mL of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water. Theresultant mixture was extracted twice with 100 mL of dichloromethaneeach and the combined organic extracts were washed twice with 10 mL ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 ml, of toluene, cooled to 0° C. and extracted with three50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40mL portions of chloroform which were combined, dried and evaporated todryness. The residue was purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) was added, the mixture was extracted with ethylacetate (3×40mL). The combined ethyl acetate layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out at room, temperature overnight.The reaction was quenched by the addition of methanol. The reactionmixture was concentrated under vacuum and to the residue dichloromethane(50 mL) was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic addmono-(4-[bis(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)esterAH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried, in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step;

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphospine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using awrist-action-shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5g, 61 mM) was added. The suspension was agitated for 2 h. The CPG wasfiltered through a sintered funnel and washed with acetonitrile,dichloromethane and ether successively. Unreacted amino groups weremasked using acetic anhydride/pyridine. The achieved loading of the CPGwas measured by taking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) was performed as described inWO 2004/065601, except, that, for the cholesteryl derivative, theoxidation step was performed using the Beaucage reagent in order tointroduce a phosphorothioate linkage at the 5′-end of the nucleic acidoligomer.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 2.

TABLE 3 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation^(a) Nucelotide(s) A, a2′-deoxy-adenosine-5′-phosphate, adenosine-5′- phosphate C, c2′-deoxy-cytidine-5′-phosphate, cytidine-5′-phosphate G, g2′-deoxy-guanosine-5′-phosphate, guanosine-5′- phosphate T, t2′-deoxy-thymidine-5′-phosphate, thymidine-5′- phosphate U, u2′-deoxy-uridine-5′-phosphate, uridine-5′- phosphate N, n any2′-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c or u) Am2′-O-methyladenosine-5′-phosphate Cm 2′-O-methylcytidine-5′-phosphate Gm2′-O-methylguanosine-5′-phosphate Tm 2′-O-methyl-thymidine-5′-phosphateUm 2′-O-methyluridine-5′-phosphate Af2′-fluoro-2′-deoxy-adenosine-5′-phosphate Cf2′-fluoro-2′-deoxy-cytidine-5′-phosphate Gf2′-fluoro-2′-deoxy-guanosine-5′-phosphate Tf2′-fluoro-2′-deoxy-thymidine-5′-phosphate Uf2′-fluoro-2-deoxy-uridine-5′-phosphate A, C, G, T, U, a, underlined:nucleoside-5′-phosphorothioate c, g, t, u am, cm, gm, tm, underlined:2-O-methyl-nucleoside-5′- um phosphorothioate ^(a)capital lettersrepresent 2′-deoxyribonucleotides (DNA), lower case letters representribonucleotides (RNA)

dsRNA Expression Vectors

In another aspect of the invention, Aha1 specific dsRNA molecules thatmodulate Aha1 gene expression activity are expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication. No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be incorporated and inherited as a transgeneintegrated into the host genome. The transgene can also be constructedto permit it to be inherited as an extrachromosomal plasmid (Gassmann,et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. In apreferred embodiment, a dsRNA is expressed as an inverted repeat joinedby a linker polynucleotide sequence such that the dsRNA has a stem andloop structure.

The recombinant dsRNA expression, vectors are generally DNA plasmids orviral vectors dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyezka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus, (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto marry different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danes and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Hubet et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10: Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector of the invention may be a eukaryotic RNA polymerase I (e.g.ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter oractin promoter or U1 snRNA promoter) or generally RNA polymerase IIIpromoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter,for example the T7 promoter, provided the expression plasmid alsoencodes T7 RNA polymerase required for transcription from a T7 promoter.The promoter can also direct transgene expression to the pancreas (see,e.g. the insulin regulatory sequence for pancreas (Bucchini et al.,1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation, by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single Aha1 gene or multiple Aha1 genes over a period, of a week ormore are also contemplated by the invention. Successful introduction ofthe vectors of the invention, into host cells can be monitored usingvarious known methods. For example, transient transfection can besignaled with a reporter, such as a fluorescent marker, such as GreenFluorescent Protein (GFP). Stable transfection. of ex vivo cells can beensured using markers that provide the transfected cell with resistanceto specific environmental, factors (e.g., antibiotics and drags), suchas hygromycin B resistance.

The Aha1 specific dsRNA molecules can also be inserted into vectors andused as gene therapy vectors for human patients. Gene therapy vectorscan be delivered to a subject by, for example, intravenous injection,local administration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

Aha1 siRNA In Vitro Screening

Single Dose Screen in HeLa and MLE 12 Cells

HeLa cells were obtained from American Type Culture Collection(Rockville, Md., cat. No. HB-8065) and cultured in Ham's F12 (BiochromAG, Berlin, Germany cat. No. FG0815) supplemented to contain 10% fetalcalf serum (FCS) (Biochrom AG, Berlin, Germany, cat. No. S0115),Penicillin 100 U/ml, Streptomycin 100 μg/ml (Biochrom AG, Berlin,Germany, cat. No. A2213) at 37° C. in an atmosphere with 5% CO₂ in ahumidified incubator (Heraeus HERAcell, Kendro Laboratory Products,Langenselbold, Germany).

MLE 12 cells were obtained from American Type Culture Collection(Rockville, Md., cat. No. CRL-2110) and cultured in HITES Medium (1:1mix Dulbecco's MEM (Biochrom AG, Berlin, Germany, cat. No: F0435)+Ham'sF12 (Biochrom AG, Berlin, Germany, cat No: FG0815)) supplemented tocontain 2% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, cat.No. S0115), Penicillin 100 U/ml, Streptomycin 100 μg/ml (Biochrom AG,Berlin, Germany, cat. No. A2213), 4 mM L-Glutamin (Biochrom AG, Berlin,Germany, cat. No: K0282), 1× Insulin/Transferrin/Na-Selenit (Gibco:51500-056), 10 nM Hydrocortisone (Sigma Munich, Germany, cat. No:H6909), 10 nM β-Estradiol (Sigma Munich, Germany, cat No: E2257), and 10mM; HEPES (USB Europe GmBH, Staufen, Germany cat No.: 16926) at 37° C.in an atmosphere with 5% CO₂ in a humidified incubator (HeraeusHERAcell, Kendro Laboratory Products, Langenselbold, Germany).

Transfection and mRNA Quantification

For transfection with siRNA, HeLa and MLE12 cells were seeded at adensity of 2.0×10⁴ cells/well in 96-well plates and transfecteddirectly. Transfection of siRNA (30 nM) was carried out withlipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No.11068-019) as described by the manufacturer. 24 hours after transfectioncells were lysed and Aha1 mRNA levels were quantified with theQuantigene Explore Kit (Genosprectra, Dumbarton Circle Fremont, USA,eat. No. QG-000-02) according to the manufacturer's protocol, Aha1 mRNAlevels were normalized to GAPDH mRNA. Readings were obtained inquadruplicates for each siRNA. siRNA duplexes unrelated to Aha1 genewere used as control. The activity of a given Aha1 specific siRNA duplexwas expressed as percent Aha1 mRNA concentration in treated cellsrelative to Aha1 mRNA concentration in cells treated with the controlsiRNA duplex.

TABLE 4 Probe sequences used with Quantigene Explore Kit (Genospectra)in quantification of Homo Sapiens (hs) Aha1 FPL Name Function SequenceSEQ ID NO: hsAha1 001 CE GATGTAAATTCCCATTGCTTCTCTTTTTTCTCTTGGAAAGAAAGT185 hsAha1 002 CE TGAACTCTGTTTTGAGGGTGCTTTTTTCTCTTGGAAAGAAAGT 186 hsAha1003 CE GGGTCTACTGACTCTCCATTCATTGTTTTTCTCTTGGAAAGAAAGT 187 hsAha1 004 CECCTTGCGCTCCTCAGTTTTCTTTTTCTCTTGGAAAGAAAGT 188 hsAha1 005 CEGGTTTTTGAAGGAGCAGGCTTAGTTTTTCTCTTGGAAAGAAAGT 189 hsAha1 006 LEACGCTGTTTTCATCAGACAAATTTTTTTAGGCATAGGACCCGTGTCT 190 hsAha1 007 LEGCTCACACTAATCTCCACTTCATCCTTTTTAGGCATAGGACCCGTGTCT 191 hsAha1 008 LETCATTAAGGCCACGAGATTTGTTTTTTAGGCATAGGACCCGTGTCT 192 hsAha1 009 LETAGGTAAGATCATGCCCTGGGTTTTTAGGCATAGGACCCGTGTCT 193 hsAha1 010 LEACTCCAACAGGTCTGGCCTGTTTTTAGGCATAGGACCCGTGTCT 194 hsAha1 011 BLGTCAGGCTCATCTTTGGCAAG 195 hsAha1 012 BL TAGAAGTTTCACCCCTTCTTCCT 196hsAha1 013 BL AGTGCTGGCTGCCCCACT 197

TABLE 5 Probe sequences used with Quantigene Explore Kit (Genospectra)in quantification of Mus musculus (mm) Aha1 FPL Name Function SequenceSEQ ID NO: mmAhsa 1001 CE CTCGAACGGCCAGGAACATTTTTCTCTTGGAAAGAAAGT 198mmAhsa 1002 CE GCACTTGCCCTCTTCATTTTCTATTTTTCTCTTGGAAAGAAAGT 199 mmAhsa1003 CE TTGATGGATGCCTCCCCATTTTTTCTCTTGGAAAGAAAGT 200 mmAhsa 1004 CEAACTCTGTCTTGAGGGTGCTGATTTTTTCTCTTGGAAAGAAAGT 201 mmAhsa 1005 CETTTGGCCTGGCTTTTTGAATTTTTCTCTTGGAAAGAAAGT 202 mmAhsa 1006 LECAAGCTTGTTCACTTCGGTCACCTCTTTTTAGGCATAGGACCCGTGTCT 203 mmAhsa 1007 LECCTGACTTAGAGGTACCTGTCCAGTTTTTTTAGGCATAGGACCCGTGTCT 204 mmAhsa 1008 LEGATTTCCACATGTCCTTTGTACTGCACTTTTTTAGGCATAGGACCCGTGTCT 205 mmAhsa 1009 LEATTTTCATCAGACAAATTGGGTTTTTAGGCATAGGACCCGTGTCT 206 mmAhsa 1010 LETAATCTCCACTTCATCCACGCTTTTTTAGGCATAGGACCCGTGTCT 207 mmAhsa 1011 LETTTCACCCCGTCTTCCTTCATTTTTAGGCATAGGACCCGTGTCT 208 mmAhsa 1012 LEACTGTGGGCAAGATCATGCCCTGAGTATTTTTAGGCATAGGACCCGTGTCT 209 mmAhsa 1013 BLAAGATAAGTTTGCCTTTCCTGTTG 210 mmAhsa 1014 BL CAGTTTGATGGTCCACTCATAGAAG211 mmAhsa 1015 BL CATCTTTGGCAAGGCTCACAC 212 mmAhsa 1016 BLTTAAGGCCACGAGATTTGTGTCAGGCT 213 mmAhsa 1017 BLGTAAATTCCCACTGCTTCTCTCAGAAG 214 mmAhsa 1018 BL CACTGGATCTACTGACTCTCCATTC215 mmAhsa 1019 BL CTCAGTCTTTAGTGCTGGCTGGCC 216 mmAhsa 1020 BLGGAGCAGACTTAGCCTTGCAAGT 217

Dose-Response Curves in HeLa Cells

Transfection and mRNA quantification: For transfection with siRNA, HeLacells were seeded at a density of 2.0×10⁴ cells/well in 96-well platesand transfected directly. Transfection of siRNA was carried out withlipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No.11668-019) as described by the manufacturer. siRNAs were concentratedfrom 30 nM in 3 fold dilutions to 14 pM, 24 hours after transfectionHeLa cells were lysed and Aha1 mRNA levels were quantified with theQuantigene Explore Kit (Genosprectra, Dumbarton Circle Fremont, USA, catNo. QG000-02) according to the protocol. Aha1 mRNA levels werenormalized to GAP-DH mRNA. For each siRNA four individual datapointswere collected, siRNA duplexes unrelated to Aha1 gene were used, ascontrol. The activity of a given Aha1 specific siRNA duplex wasexpressed as percent Aha1 mRNA concentration in treated cells relativeto Aha1 mRNA concentration in cells treated with the control siRNAduplex. XL-fit was, used to calculate IC₅₀ values.

Table 6 provides values for inhibition of Aha1 expression using variousdsRNA molecules of the invention.

TABLE 6 Residual Aha1 mRNA in % of control in HeLa and MLE12 cellstreated with 30 nM solutions of various RNAi agents specific for Aha1,and IC₅₀ for selected RNAi agents determined in HeLa cells HeLa cells,IC₅₀ in MLE12 cells, Duplex residual HeLa cells residual identifier mRNA[%] [nM] mRNA [%] AL-DP-7299 19 ± 3  105 ± 17  AL-DP-7300 7 ± 2 94 ± 13AL-DP-7301 3 ± 1 0.035 14 ± 3  AL-DP-7302 17 ± 5  61 ± 16 AL-DP-7303 5 ±2 23 ± 5  AL-DP-7304 7 ± 3 30 ± 7  AL-DP-7305 6 ± 2 26 ± 6  AL-DP-730627 ± 8  45 ± 11 AL-DP-7307 16 ± 6  1.1 27 ± 8  AL-DP-7308 13 ± 5  0.2120 ± 7  AL-DP-7309 10 ± 3  0.36 22 ± 8  AL-DP-7310 51 ± 13 59 ± 13AL-DP-7311 4 ± 3 0.07 16 ± 4  AL-DP-7312 14 ± 3  40 ± 8  AL-DP-7313 63 ±14 80 ± 10 AL-DP-7314 97 ± 21 88 ± 10 AL-DP-7315 76 ± 24 77 ± 10AL-DP-7316 7 ± 2 0.34 23 ± 6  AL-DP-7317 11 ± 3  44 ± 12 AL-DP-7318 4 ±2 0.29 16 ± 3  AL-DP-7319 38 ± 7  73 ± 21 AL-DP-7320 16 ± 4  0.07 15 ±5  AL-DP-7321 130 ± 36  81 ± 16 AL-DP-7322 4 ± 2 0.045 10 ± 3 AL-DP-7323 24 ± 6  55 ± 11 AL-DP-7324 3 ± 2 0.089 12 ± 4  AL-DP-7325 5 ±2 0.3 12 ± 4  AL-DP-7326 3 ± 1 0.27 19 ± 7  AL-DP-7327 3 ± 1 0.08 13 ±7  AL-DP-7328 49 ± 14 67 ± 10 AL-DP-7329 6 ± 2 0.2 18 ± 5  AL-DP-7330 64± 19 78 ± 10 AL-DP-7331 5 ± 2 0.55 13 ± 5  AL-DP-7332 95 ± 20 82 ± 15AL-DP-7333 2 ± 1 0.27 9 ± 3 AL-DP-7334 94 ± 17 83 ± 19 AL-DP-7335 11 ±5  57 ± 11 AL-DP-7336 22 ± 4  63 ± 12 AL-DP-7337 6 ± 2 0.29 20 ± 5 AL-DP-7338 39 ± 6  56 ± 10 AL-DP-7339 10 ± 1  35 ± 6  AL-DP-7340 8 ± 20.61 19 ± 6  AL-DP-7341 17 ± 4  55 ± 16 AL-DP-7342 6 ± 4 0.5 15 ± 3 AL-DP-7343 26 ± 4  103 ± 19  AL-DP-7344 5 ± 2 38 ± 11 AL-DP-7345 53 ± 2263 ± 15 AL-DP-7346 22 ± 4  44 ± 11 AL-DP-9250 4 ± 1 AL-DP-9251 51 ± 9 AL-DP-9252 19 ± 2  AL-DP-9253 11 ± 1  AL-DP-9254 7 ± 1 AL-DP-9255 5 ± 0AL-DP-9256 5 ± 0 AL-DP-9257 7 ± 0 AL-DP-9258 9 ± 1 AL-DP-9259 7 ± 1AL-DP-9260 15 ± 3  AL-DP-9261 21 ± 2  AL-DP-9262 24 ± 4  AL-DP-9263 25 ±6  AL-DP-9264 7 ± 2 AL-DP-9265 8 ± 1 AL-DP-9266 11 ± 2  AL-DP-9267 45 ±4  AL-DP-9268 9 ± 1 AL-DP-9269 5 ± 1 AL-DP-9270 6 ± 1 AL-DP-9271 6 ± 2AL-DP-9272 26 ± 8  AL-DP-9273 11 ± 1  AL-DP-9274 7 ± 1 AL-DP-9275 8 ± 1AL-DP-9276 4 ± 1 AL-DP-9277 10 ± 1  AL-DP-9278 2 ± 0 AL-DP-9279 3 ± 0AL-DP-9280 12 ± 1  AL-DP-9281 8 ± 2 AL-DP-9282 3 ± 0 AL-DP-9283 6 ± 1AL-DP-9284 39 ± 2  AL-DP-9285 4 ± 1 AL-DP-9286 61 ± 11 AL-DP-9287 3 ± 1AL-DP-9288 27 ± 5  AL-DP-9289 6 ± 1

In summary, AL-DP-7301, AL-DP-7303, AL-DP-7318, AL-DP-7320, AL-DP-7322,AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329, AL-DP-7331,AL-DP-7333, AL-DP-7340, and AL-DP-7342 inhibited Aha1 expression by atleast 80% in both HeLa and MLE12 cells, AL-DP-7303, AL-DP-7305,AL-DP-7307, AL-DP-7309, AL-DP-7316, and AL-DP-7337 inhibited Aha1expression by at least 80% in HeLa cells and by at least 70% in MLE12cells, AL-DP-7304, AL-DP-7312, AL-DP-7339, and AL-DP-7344 inhibited Aha1expression by at least 80% in HeLa cells and by at least 60% in MLE12cells, AL-DP-7306, AL-DP-7317, and AL-DP-7346 inhibited Aha1 expressionby at least 70% in HeLa cells and by at least 50% in MLE 12 cells,AL-DP-7310, AL-DP-7323, AL-DP-7335, AL-DP-7338, and AL-DP-7341 inhibitedAha1 expression by at least 40% in both HeLa and MLE12 cells, andAL-DP-7302, AL-DP-7315, AL-DP-7328, AL-DP-7330, AL-DP-7336, andAL-DP-7345, inhibited Aha1 expression by at least 20% in both HeLa andMLE12 cells.

In addition, AL-DP-9250, AL-DP-9252, AL-DP-9253, AL-DP-9254, AL-DP-9255,AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9264,AL-DP-9265, AL-DP-9266, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276, AL-DP-9277, AL-DP-9279,AL-DP-9280, AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-DP-9285, AL-DP-9287,and AL-DP-9289 inhibited Aha1 expression by at least 80% in HeLa cells,AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9272, and AL-DP-9288 inhibitedAha1 expression by at least 70% in HeLa cells, AL-DP-9263 inhibited Aha1expression by at least 60% in HeLa cells, AL-DP-9267 inhibited Aha1expression by at least 50% in HeLa cells, AL-DP-9251 inhibited Aha1expression by at least 40% in HeLa cells, and AL-DP-9286 inhibited Aha1expression by at least 3.0% in HeLa cells.

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting theexpression of a human Aha gene, wherein the dsRNA consists of SEQ IDNO:51 and SEQ ID NO:52.
 2. The dsRNA of claim 1, wherein said Aha geneis an Aha1 gene.
 3. The dsRNA of claim 1, wherein, upon contact with acell expressing said Aha gene, the dsRNA inhibits mRNA expression ofsaid Aha gene in said cell by at least 20% compared to a control siRNAduplex unrelated to said Aha gene.
 4. The dsRNA of claim 3, wherein saidat least 20% inhibition of mRNA expression of said Aha gene is effectedin HeLa and/or MLE12 cells.
 5. The dsRNA of claim 1, wherein said dsRNA,upon contact with a cell expressing said Aha, inhibits mRNA expressionof said Aha gene by at least 25% compared to a control siRNA duplexunrelated to said Aha gene.
 6. The dsRNA of claim 1, wherein said dsRNA,upon contact with a cell expressing said Aha, inhibits mRNA expressionof said Aha gene by at least 40% compared to a control siRNA duplexunrelated to said Aha gene.
 7. A cell comprising the dsRNA of claim 1.8. A vector for inhibiting the expression of an Aha gene in a cell, saidvector encoding the dsRNA of claim
 1. 9. A cell comprising the vector ofclaim
 8. 10. A pharmaceutical composition for inhibiting the expressionof an Aha gene, comprising the dsRNA of claim 1 and a pharmaceuticallyacceptable carrier.
 11. The pharmaceutical composition of claim 10,wherein said Aha gene is an Aha1 gene.
 12. The pharmaceuticalcomposition of claim 10, wherein, upon contact with a cell expressingsaid Aha gene, the dsRNA inhibits mRNA expression of said Aha gene insaid cell by at least 20% compared to a control siRNA duplex unrelatedto said Aha gene.
 13. The pharmaceutical composition of claim 12,wherein said at least 20% inhibition of mRNA expression of said Aha geneis effected in HeLa and/or MLE12 cells.
 14. The pharmaceuticalcomposition of claim 10, wherein the carrier is a lipid carrier.